Method of treating prostatic diseases using active vitamin D analogues

ABSTRACT

The invention provides therapeutic methods for inhibiting, ameliorating or alleviating the hyperproliferative cellular activity of diseases of the prostate, e.g., prostatic cancer and prostatic hyperplasia, which includes administering to a patient in need thereof an active vitamin D analogue. Cell differentiation is promoted, induced or enhanced without causing to the patient dose-limiting hypercalcemia and hypercalciuria.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of Ser. No.08/415,488. Apr. 3, 1995, which is a continuation-in-part of Ser. No.08/119,895, Sep. 10, 1993, now U.S. Pat. No. 5,403,831, and is also acontinuation-in-part of Ser. No. 08/486,387, Jun. 7, 1995, which is acontinuation-in-part of Ser. No. 08/265,438, Jun. 24, 1994, all of whichare incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable

BACKGROUND OF THE INVENTION

[0003] This invention relates generally to a method of treatinghyperproliferative prostatic diseases, and in particular, to the use ofactive forms of vitamin D to inhibit the hyperproliferative cellularactivity of these diseases and to promote differentiation of the cells.

[0004] The prostate gland is found exclusively in male mammals and issubject to certain hyperproliferative diseases. A proliferation of basaland stroma cells of the prostate gland gives rise to benign prostatichyperplasia which is one common prostate disease. Another commonprostate disease is prostate cancer, especially prostaticadenocarcinoma. Adenocarcinoma of the prostate is the most common of thefatal pathophysiological prostate cancers, and typically involves amalignant transformation of epithelial cells in the peripheral region ofthe prostate gland. Both prostatic hyperplasia and prostate cancer havea high rate of incidence in the aging human male population.Approximately one out of every four males above the age of 55 suffersfrom a prostate disease of some form or another.

[0005] Prostate cancer is currently the second most frequent cause ofcancer death after lung cancer among American males. Mortality rates forprostate cancer increase logarithmically with age and are two timeshigher in U.S. blacks than whites. Internationally, mortality rates arehighest in U.S. blacks and in northern Europe and are lowest in Japan.It is projected that by the year 2000, a 90% increase in annualincidence of the disease and a 37% increase in annual mortality rateswill be observed. Although prostate cancer may be a relatively indolentneoplasm in the elderly, the overall decrease in life span in patientswith this disease is approximately 10 years.

[0006] Improvement in the treatment of prostate cancer has centered onearly detection. In recent years, screening tests which detect certainproteins or peptides secreted by the prostate gland, i.e., markers,(e.g, prostate-specific antigen (PSA), prostatic acid phosphatase (PAP),prostatic inhibin (PIP)), have increased the power to diagnose thisdisease in asymptomatic patients.

[0007] Treatment of prostate cancer in men under the age of 65 hasfocused on radical surgery, e.g., prostatectomy, and/or radiotherapy,but the impact of these aggressive approaches on overall survivalremains debatable. The approach to treatment of men over the age of 65historically has been more conservative, and is based on the ablation orcontrol of testosterone production. Such ablation or control is usuallyachieved by surgical castration, by administration of pituitarygonadotropin inhibitors such as estrogens or luteinizing hormonereleasing hormone (LHRH) analogues, or a combination of these treatmentmethods. Estrogens, such as diethylstilbestrol, are potent inhibitors ofthe release from the pituitary gland of luteinizing hormone (LH), thegonadotropin that regulates testosterone production, and consequently,estrogen administration can cause a fall in testosterone to castrationlevels. Maximum suppression of plasma testosterone is typically achievedby a dosage of 3 mg/day of diethylstilbestrol. Other estrogens such asconjugated estrogens are about as equally effective in the lowering ofthe plasma level as diethylstilbestrol. However, diethylstilbestrol hasa poor cardiovascular profile, and death from cardiovascular disease isnot uncommon in patients treated with large doses of diethylstilbestrol.Thus, while dosages of up to 3 mg/day of diethylstilbestrol aretypically safe, this treatment regime is not indicated for men withpreexisting cardiovascular disease.

[0008] Prostatic carcinoma often metastasizes to the pelvis and lumbarvertebrae, causing bone loss and associated pain. Hormone manipulationoften may result in significant palliation of metastatic prostatecancer, with improvement of bone pain and other disease-associatedsymptoms. Androgen ablation is, thus, also a major adjunctive therapy inadvanced metastatic prostate cancer.

[0009] Despite initial improvement on hormonal treatment, a majority ofpatients with locally unresectable or metastatic disease will eventuallyfail to respond to further hormonal therapies. A recent study suggeststhat human prostate cancer cells may cycle between beingandrogen-independent and androgen-dependent. Such cycling may accountfor the return of the cancer after initial improvement. In this largegroup of patients, other forms of treatment, unfortunately, are far lesseffective. Radiotherapy often may relieve the symptoms of bone pain, butis not curative. Over time, the disease will progress with a fataloutcome.

[0010] As noted hereinabove, prostatic hyperplasia is another commonhyperproliferative disease of the prostate gland. The disorder affectsmen over the age of 45 and increases in frequency with age. Prostatichyperplasia begins in the periurethral region as a localizedproliferation and progresses to compress the remaining normal gland. Thehyperplasia can compress and obstruct the urethra. Treatment includessurgery, and administration of pituitary gonadotropin inhibitors and/or5α-reductase enzyme inhibitors.

[0011] In another area of physiology and biochemistry, the vitamin Darea, extensive research during the past two decades has establishedimportant biologic roles for vitamin D apart from its classic role inbone and mineral metabolism. Specific nuclear receptors for1α,25-dihydroxyvitamin D₃, the hormonally active form of vitamin D, arepresent in cells from diverse organs not involved in calciumhomeostasis. For example, Miller et al., 52 Cancer Res. (1992) 515-520,have demonstrated specific, biologically active receptors for1α,25-dihydroxyvitamin D₃ in the human prostatic carcinoma cell line,LNCaP.

[0012] It has been reported that certain vitamin D compounds andanalogues are potent inhibitors of malignant cell proliferation and areinducers/stimulators of cell differentiation. For example, U.S. Pat. No.4,391,802 issued to Suda et al. discloses that 1α-hydroxyvitamin Dcompounds, specifically 1α,25-dihydroxyvitamin D₃ and 1α-hydroxyvitaminD₃, possess potent antileukemic activity by virtue of inducing thedifferentiation of malignant cells (specifically leukemia cells) tononmalignant macrophages (monocytes), and are useful in the treatment ofleukemia. Antiproliferative and differentiating actions of1α,25-dihydroxyvitamin D₃ and other vitamin D₃ analogues have beenreported with respect to prostate cancer cell lines. More recently, anassociation between vitamin D receptor gene polymorphism and prostatecancer risk has been reported, suggesting that vitamin D receptors mayhave a role in the development, and possible treatment, of prostatecancer.

[0013] These previous studies have focused exclusively on vitamin D₃compounds. Even though these compounds may indeed be highly effective inpromoting differentiation in malignant cells in culture, their practicaluse in differentiation therapy as anticancer agents is severely limitedbecause of their equally high potency as agents affecting calciummetabolism. At the levels required in vivo for effective use as, forexample, antileukemic agents, these same compounds can induce markedlyelevated and potentially dangerous blood calcium levels by virtue oftheir inherent calcemic activity. That is, the clinical use of1α,25-dihydroxyvitamin D₃ and other vitamin D₃ analogues as anticanceragents is precluded, or severely limited, by the risk of hypercalcemia.This indicates a need for compounds with greater specific activity andselectivity of action, i.e., vitamin D compounds with antiproliferativeand differentiating effects but which have less calcemic activity. Theneed for such compounds is no greater than in the treatment ofneoplastic and hyperplastic prostatic diseases.

BRIEF SUMMARY OF THE INVENTION

[0014] The present invention provides a method of treating prostaticdisease conditions such as those characterized by hyperproliferativecell growth and/or abnormal cell differentiation, e.g., prostate cancerand prostatic hyperplasia. The method includes use of active vitamin Dcompounds to inhibit abnormal cell growth and promote celldifferentiation.

[0015] The foregoing, and other advantages of the present invention, arerealized in one aspect thereof in a method of inhibiting thehyperproliferative activity of human neoplastic or hyperplastic cells,comprising treating the cells with an effective amount of a1α-hydroxyvitamin D compound having a hydrocarbon moiety substituted atthe C-24 position on the sidechain of the molecule. The treating stepincludes inhibiting proliferation of, and inducing and enhancingdifferentiation in such prostatic cells.

[0016] The 1α-hydroxyvitamin D compound is an active vitamin D and issuitably represented by the formula (I) described hereinafter. Preferredamong the compounds of formula (I), are 1α,24-dihydroxyvitamin D₂,1α,24-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₄,1α,25-dihydroxyvitamin D₂, 1α-hydroxyvitamin D₂ and 1α-hydroxyvitaminD₄.

[0017] The effective or therapeutic amount of the 1α-hydroxyvitamin Dcompound administrable in accordance with the present invention topatients in need on a daily basis per kilogram of body weight rangesfrom 0.01 pg/kg/day to 2.0 pg/kg/day.

[0018] In another aspect, the invention is a method of treating humanprostate cancer, comprising administering to a male subject who hasprostate cancer an effective amount of an active vitamin D compoundwhich has, or attains through metabolism in vivo, a vitamin D receptor(VDR) binding affinity substantially equivalent to the binding affinityof 1α,25-dihydroxyvitamin D₃ and a hypercalcemia risk substantiallylower than that of 1α,25-dihydroxyvitamin D₃, to decrease or stabilizethe cellular abnormal proliferative activity of the cancer.

[0019] For treatment for prostate conditions in accordance with thepresent invention, the active vitamin D is suitably administered aloneas an active ingredient, i.e., as a first anticancer agent, in apharmaceutical composition, or in a mixture including a secondanticancer agent, an androgen abalation agent, a 5α-reductase inhibitoror combinations thereof.

[0020] In another aspect, the invention is a pharmaceutical compositionwhich includes a first anticancer agent which is an active vitamin Dcompound; an agent selected from the group consisting of (i) a secondanticancer agent, (ii) a bone agent, (iii) an androgen ablation agentand (iv) a 5α-reductase inhibitor and combinations thereof; and aphysiologically acceptable carrier.

[0021] Other advantages and a fuller appreciation of specificadaptations, compositional variations, and physical attributes will begained upon an examination of the following detailed description ofpreferred embodiments, taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWING(S)

[0022] Not Applicable

DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides an effective method for thetreatment of neoplastic and hyperplastic diseases. Particularly, thepresent invention relates to therapeutic methods for inhibiting,ameliorating or alleviating the hyperproliferative cellular activity ofdiseases of the prostate, e.g., prostatic cancer and prostatichyperplasia, and inducing, enhancing or promoting cell differentiationin the diseased cells. The present invention provides a novel treatmentof a patient suffering from a hyperproliferative disease such asprostatic cancer or prostatic hyperplasia with an active vitamin Danalogue having a hydrocarbon moiety substituted at the C-24 position ofthe sidechain of the molecule. Preferably, the active vitamin D analogueis a 1α-hydroxyvitamin D compound and is suitably represented by formula(I) as described hereinbelow. The active vitamin D analogue is providedto the patient without causing dose-limiting hypercalcemia andhypercalciuria, i.e., unphysiologically high and deleterious bloodcalcium levels and urine calcium levels, respectively. These attributesare achieved through specific chemical properties of the compounds offormula (I) described.

[0024] In accordance with the present invention, when effective amountsof the analogues of formula (I) are administered to patients withprostatic cancer or prostatic hyperplasia, the proliferative activity ofthe abnormal prostatic cells is inhibited or alleviated, and celldifferentiation is induced, promoted or enhanced, with significantlyless hypercalcemia and hypercalciuria than is observed after the sameamount of activated vitamin D₃ is administered in previously knownformulations. Thus, the compounds of formula (I) have an improvedtherapeutic index relative to active forms of vitamin D₃ analogues.

[0025] It is known that vitamin D₃ must be hydroxylated in the C-1 andC-25 positions before it is activated, i.e., before it will produce abiological response. A similar metabolism appears to be required toactivate other forms of vitamin D, e.g., vitamin D₂ and vitamin D₄.Therefore, as used herein, the term “activated vitamin D” or “activevitamin D” is intended to refer to a vitamin D compound or analogue thathas been hydroxylated in at least the C-1 position of the A ring of themolecule and either the compound itself or its metabolites in the caseof a prodrug, such as 1α-hydroxyvitamin D₂, binds the vitamin D receptor(VDR). Vitamin D compounds which are hydroxylated only in the C-1position are referred to herein as “prodrugs.” Such compounds undergofurther hydroxylation in vivo and their metabolites bind the VDR.

[0026] Also, as used herein, the term “lower” as a modifier for alkyl,alkenyl acyl, or cycloalkyl is meant to refer to a straight or branched,saturated or unsaturated hydrocarbon radical having 1 to 4 carbon atoms.Specific examples of such hydrocarbon radicals are methyl, ethyl,propyl, isopropyl, butyl, isobutyl, t-butyl, ethenyl, propenyl, butenyl,isobutenyl, isopropenyl, formyl, acetyl, propionyl, butyryl orcyclopropyl. The term “aromatic acyl” is meant to refer to aunsubstituted or substituted benzoyl group.

[0027] As used herein, the term “hydrocarbon moiety” refers to a loweralkyl, a lower alkenyl, a lower acyl group or a lower cycloalkyl, i.e.,a straight or branched, saturated or unsaturated C₁-C₄ hydrocarbonradial.

[0028] The compound in accordance with the present invention is anactive vitamin D compound provided that such compound has a hydrocarbonmoiety at the C-24 position, e.g., a lower alkyl, alkenyl or acyl groupat the C-24 position. Further, the active vitamin D in accordance withthe present invention may have an unsaturated sidechain, e.g., there issuitably a double bond between C-22 and C-23, between C-25 and C-26 orbetween C-26 and C-27.

[0029] The 1α-hydroxyvitamin D of the present invention preferably hasthe general formula described in formula (I)

[0030] wherein B and C each are hydrogen or a carbon-carbon bond, thusforming a double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl, or taken together withthe carbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, and X² is hydrogen or hydroxyl,or, may be taken with R¹ or R², to constitute a double bond.

[0031] The 1α-hydroxyvitamin D compounds of formula (I) of the presentinvention are those that have effective antiproliferative and celldifferentiation activity (i.e., reversal of malignant transformation),particularly with respect to cells of prostatic diseases, e.g.,prostatic cancer and prostatic hyperplasia, but have a lower tendency orinability to cause the undesired side effects of hypercalcemia and/orhypercalciuria. In other words, the compounds of formula (I) can beadministered at dosages that allow them to act as antiproliferativeagents and cell differentiation agents when exposed to malignant orother hyperproliferative cells without significantly altering calciummetabolism. This selectivity and specificity of action makes the1α-hydroxyvitamin D compounds of formula (I) useful and preferred agentsfor safely inhibiting hyperproliferation and promoting malignant orhyperplastic cell differentiation. The 1α-hydroxyvitamin D compounds ofthe present invention, thus, overcome the shortcomings of the knownactive vitamin D₃ compounds described above, and can be consideredpreferred agents for the control and treatment of malignant diseasessuch as prostate cancer as well as benign prostatic hyperplasia.

[0032] Preferred among the active vitamin D compounds of formula (I)are: 1α,24-dihydroxyvitamin D₂, 1α,24-dihydroxyvitamin D₄,1α,25dihydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitaminD₂, and 1α-hydroxyvitamin D₄. Among those compounds of formula (I) thathave a chiral center in the sidechain, such as at C-24, it is understoodthat both epimers (e.g., R and S) and the racemic mixture are within thescope of the present invention.

[0033] Thus, the present invention provides a method of treatingmalignant prostatic cells as well as other hyperproliferative prostaticcells, (i.e., inhibiting their hyperproliferative activity and/orinducing and enhancing their differentiation) with an effective amountof a compound of formula (I). The effective dosage amount on a dailybasis per kilogram of body weight of the patient ranges from about 0.01pg/kg/day to about 2.0 μg/kg/day.

[0034] The compounds of formula (I) are valuable for the treatment ofprostate cancer and prostatic hyperplasia in a patient sufferingtherefrom. In particular, the invention is a method for treating apatient suffering from the hyperproliferative cellular effects ofprostate cancer and prostatic hyperplasia by administering to thepatient a therapeutically effective amount of a compound of formula (I),which is preferably 1α,24-dihydroxyvitamin D₂, 1α,24-dihydroxyvitaminD₄, 1α,25-dihydroxyvitamin D₂, 1α,25-dihydroxyvitamin D₄,1α-hydroxyvitamin D₂, and 1α-hydroxyvitamin D₄.

[0035] The compounds of formula (I) can be prepared as described, e.g.,in U.S. Pat. No. 5,448,120 issued to Knutson et al., U.S. Pat. No.4,554,106 issued to DeLuca et al., and Strugnell et al., 310 Biochem. J.(1995) pp. 233-241, all of which are incorporated herein by reference.

[0036] The biopotencies of the compounds of formula (I) have beenstudied and compared to that of 1α,25-dihydroxyvitamin D₃, the activehormonal form of vitamin D and the standard against which all vitamin Dcompounds and analogues are measured. For example, it has been foundthat the vitamin D receptor (VDR) binding affinities of the compounds offormula (I), or their active metabolites, are substantially equivalentto (i.e., equal to or up to 3 times weaker than) the affinity of1α,25-dihydroxyvitamin D₃. Such receptor binding affinities areindicative of potent biological activity.

[0037] At the same time, it has been found that compounds of formula (I)are significantly less toxic than their corresponding vitamin D₃analogues. For example, in parent co-pending application, Ser. No.08/265,438, the disclosure of which is incorporated herein by reference,the LD₅₀ for 1α-hydroxyvitamin D₄ was found to be 1.0 mg/kg in males and3.0 mg/kg in females, i.e., substantially less toxic than1α-hydroxyvitamin D₃ (LD₅₀˜0.2 mg/kg). Further, in the parent U.S. Pat.No. 5,403,831, and its grandparent U.S. Pat. No. 5,104,864, both ofwhich are incorporated herein by reference, it has been shown that1α-hydroxyvitamin D₂ has the same biopotency as 1α-hydroxyvitamin D₃ and1α,25-dihydroxyvitamin D₃ but is much less toxic. Even dosages up to 10μg/day of 1α-hydroxyvitamin D₂ in women with postmenopausal osteoporosiselicited only mild hypercalciuria (U.Ca>300 mg/24 hrs), and no markedhypercalcemia (S. Ca>11.0 mg/dL) solely due to 1α-hydroxyvitamin D₂ wasevident. Additionally, the compound did not adversely affect kidneyfunction, as determined by creatinine clearance and BUN; nor did itincrease urinary excretion of hydroxyproline, indicating the absence ofany stimulatory effect on bone resorption. Administration of1α-hydroxyvitamin D₂ to healthy adult males in dosages up to 8 μg/dayshowed no clinically significant hypercalcemia or other adverse effects.

[0038] The compounds of formula (I) are useful as active compounds inpharmaceutical compositions having reduced side effects and low toxicityas compared with the known analogues of active forms of vitamin D₃.

[0039] The pharmacologically active compounds of this invention can beprocessed in accordance with conventional methods of pharmacy to producemedicinal agents for administration to patients, e.g., mammals includinghumans. For example, the compounds of formula (I) can be employed inadmixtures with conventional excipients, e.g., pharmaceuticallyacceptable carrier substances suitable for enteral (e.g., oral) orparenteral application which do not deleteriously react with the activecompounds.

[0040] Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions, alcohols, gum arabic, vegetable oils(e.g., corn oil, cottonseed oil, peanut oil, olive oil, coconut oil),fish liver oils, oily esters such as Polysorbate 80, polyethyleneglycols, gelatin, carbohydrates (e.g., lactose, amylose or starch),magnesium stearate, talc, silicic acid, viscous paraffin, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, polyvinyl pyrrolidone, etc.

[0041] The pharmaceutical preparations can be sterilized and, ifdesired, be mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, coloring, flavoring and/or one ormore other active agents.

[0042] For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solution, as well assuspensions, emulsions, or implants, including suppositories. Ampulesare convenient unit dosages.

[0043] For enteral application, particularly suitable are tablets,dragees, liquids, drops, lozenges, powders, or capsules. A syrup,elixir, or the like can be used if a sweetened vehicle is desired.

[0044] For rectal administration, compounds are formed into apharmaceutical composition containing a suppository base such as cacaooil or other triglycerides. To prolong storage life, the compositionadvantageously includes an antioxidant such as ascorbic acid, butylatedhydroxyanisole or hydroquinone.

[0045] Oral administration of the pharmaceutical compositions of thepresent invention is preferred. The dosage of the compounds for thetreatment of prostatic cancer or hyperplasia according to this inventiongenerally is about 0.01 to about 2.0 μg/kg/day, preferably about 0.01 toabout 1.0 μg/kg/day. Generally, the compounds of this invention aredispensed by unit dosage form in a pharmaceutically acceptable carrier.

[0046] For treatment of prostate cancer, the parenteral dosage of thecompounds of formula (I) is about 0.01 μg/kg/day to about 1.0 μg/kg/day.

[0047] It will be appreciated that the actual preferred amounts ofactive compound in a specific case will vary according to the efficacyof the specific compound employed, the particular compositionsformulated, the mode of application, and the particular situs andorganism being treated. For example, the specific dose for a particularpatient depends on age, body weight, general state of health, on diet,on the timing and mode of administration, on the rate of excretion, andon medicaments used in combination and the severity of the particulardisorder to which the therapy is applied. Dosages for a given host canbe determined using conventional considerations, e.g., by customarycomparison of the differential activities of the subject compounds andof a known agent, such as by means of an appropriate conventionalpharmacological protocol.

[0048] Also included within the scope of the present invention is theco-administration of a compound of formula (I) with known androgencontrol or ablation or testosterone level-lowering agents such asestrogens (e.g., diethylstilbestrol), LHRH analogues, 5α-reductaseenzyme inhibitors such as finasteride, antiestrogens (e.g., Tamoxifen™),and antiandrogens (e.g., flutamide). (See, e.g., U.S. Pat. No.5,372,996, incorporated herein by reference.) It is anticipated that asymbiotic effect is obtainable with these various combinations, and willprovide an increased therapeutic effect. Also, there is the potential toprovide therapy wherein the adverse side effects with some of theseagents, e.g., the deleterious cardiovascular effects of estrogens, areconsiderably reduced compared to when these agents are used alone inlarger dosages. Possible dose ranges of these co-administered androgencontrol or testosterone level-lowering agents are about 0.01 to 0.20μg/kg/day.

[0049] Further, included within the scope of the present invention isthe co-administration of the active vitamin D of formula (I) with asecond anticancer agent, e.g., a cytotoxic agent, particularly inmetastatic prostate cancer wherein relapse has occurred followinghormonal treatment. Such agents may suitably include estramustinephosphate, prednimustine, cisplatin, 5-fluoro-uracil, melphalan,hydroxyurea, mitomycin, idarubicin, methotrexate, adriamycin anddaunomycin. It is anticipated that an active vitamin D of formula (I)used in combination with various anticancer drugs can give rise to asignificantly enhanced cytotoxic effect on cancerous cells, thusproviding an increased therapeutic effect. Specifically, as asignificantly increased growth-inhibitory effect is obtained with theabove disclosed combinations utilizing lower concentrations of theanticancer drugs compared to the treatment regimes in which the drugsare used alone, there is the potential to provide therapy whereinadverse side effects associated with the anticancer drugs areconsiderably reduced than normally observed with the anticancer drugsused alone in larger doses. Possible dose ranges of theseco-administered second anticancer agents are about 0.1 to 1 μg/kg/day.

[0050] Also included within the scope of the present invention is theco-administration of effective dosages of the analogue of formula (I) inconjunction with administration of hormones or other agents, e.g.,estrogens, which are known to ameliorate bone diseases or disorders. Asnoted above, prostate cancer often metastasizes to bone, causing boneloss and associated pain. Such bone agents may include conjugatedestrogens or their equivalents, calcitonin, bisphosphonates, calciumsupplements, cobalamin, pertussis toxin and boron. Possible dose rangesfor these co-administered bone agents are provided in Table 1. TABLE 1Possible Oral Dose Ranges for Various Bone Agents Co-Administered With1α-Hydroxyvitamin D of Formula (I) Dose Ranges Agent Broad PreferredMost Preferred Conjugated Estrogens or 0.3-5.0 0.4-2.4 0.6-1.2Equivalent (mg/day) Sodium Fluoride (mg/day)  5-150 30-75 40-60Calcitonin (IU/day)  5-800  25-500  50-200 Bisphosphonates (mg/day)0.5-20   1-15  5-10 Calcium Supplements  250-2500  500-1500  750-1000(mg/day) Cobalamin (μg/day)  5-200  20-100 30-50 Pertussis Toxin(mg/day)   0.1-2000  10-1500  100-1000 Boron (mg/day)  0.10-3000  1-250 2-100

[0051] Antiestrogens, such as Tamoxifen™, are also known bone agents andmay be suitably used in conjunction with the 1α-hydroxyvitamin Dcompounds of the present invention.

[0052] The present invention is further explained by the followingexamples which should not be construed by way of limiting the scope ofthe present invention.

VDR Binding Analyses EXAMPLE 1

[0053] 1α,24-dihydroxyvitamin D₂ [1α,24-(OH)₂D₂]

[0054] The affinity of 1α,24-(OH)₂D₂ for the mammalian vitamin Dreceptor (VDR) was assessed using a commercially available kit of bovinethymus VDR and standard 1,25-(OH)₂D₃ solutions from Incstar (Stillwater,Minn.). The half-maximal binding of chemically synthesized 1α,24-(OH)₂D₂was approximately 150 μg/ml whereas that of 1α,25-(OH)₂D₃ was 80 μg/mI.Thus, the 1α,24-(OH)₂D₂ had a very similar affinity for bovine thymusVDR as did 1α,25-(OH)₂D₃, indicating that 1α,24-(OH)₂D₂ has potentbiological activity.

EXAMPLE 2

[0055] 1α,24-dihydroxy vitamin D₄ [1α,24-(OH)₂D₄]

[0056] The VDR affinity binding of 1α,24-(OH)₂D₄ was investigated. The1α,24-(OH)₂D₄ was incubated with vitamin D receptor and radiolabeledtracer 1α,25-(OH)₂D₃. After incubation, the amount of radioactivitybound to the receptor was determined and compared with the amount boundafter co-incubation of unlabeled and labeled 1α,25-(OH)₂D₃. It was foundthat 50 pg/tube of 1α,24-(OH)₂D₄ was equivalent to approximately 20 pg1α,25-(OH)₂D₃.

[0057] These results show that 1α,24-(OH)₂D₄ binds slightly less tightlyto the vitamin D receptor than does 1α,25-(OH)₂D₃. Such data mean that1α,24-(OH)₂D₄ has high affinity for the VDR and significant biologicalactivity, similar to that of 1α,25-(OH)₂D₃. These data are consistentwith gene expression studies done (described below) with 1α,24-(OH)₂D₄which demonstrate that 1α,24-(OH)₂D₄ is only slightly less active thanis 1α,25-(OH)₂D₃.

[0058] These results are surprising and unexpected in view of the priorart. They are contrary to the normative wisdom in the vitamin D artregarding the very low degree of biological activity of vitamin D₄compounds.

EXAMPLE 3

[0059] 1α,24-dihydroxyvitamin D₂ [1α,24-(OH)₂D₂]

[0060] VDR binding of vitamin D compounds by prostate cells isdemonstrated using the techniques of Skowronski et al., 136Endocrinology (1995) 20-26, which is incorporated herein by reference.Prostate-derived cell lines are cultured to near confluence, washed andharvested by scraping. Cells are washed by centrifugation, and the cellpellet resuspended in a buffered salt solution containing proteaseinhibitors. The cells are disrupted by sonication while cooling on ice.The supernatant obtained from centrifuging the disrupted cells at207,000×g for 35 min at 4° C. is assayed for binding. 200 μL of solubleextract, (1-2 mg protein/ml supernatant) is incubated with a 1 nM³H-1α,25-(OH)₂D₃ and increasing concentrations of 1α,24-(OH)₂-D₂(0.01-100 nM) for 16-20 hr at 4° C. Bound and free hormones areseparated with hydroxylapatite using standard procedures. Specificbinding is calculated by subtracting nonspecific binding obtained in thepresence of a 250-fold excess of nonradioactive 1α,25-(OH)₂D₃ from thetotal binding measured. The results demonstrate that 1α,24-(OH)₂D₂ hasstrong affinity for prostate VDR, indicating that 1α,24-(OH)₂D₂ haspotent biological activity in respect of prostate cells.

EXAMPLE 4

[0061] 1α,24-dihydroxy vitamin D₄ [1α,24-(OH)₂D₄]

[0062] The procedure of Example 3 is repeated using the active vitamin Danalogue 1α,24-(OH)₂D₄, and the specific binding is determined. Theresults demonstrate that 1α,24-(OH)₂D₄ has strong affinity for prostateVDR, indicating that 1α,24-(OH)₂D₄ has potent biological activity inrespect of prostate cells.

EXAMPLE 5

[0063] 1α,25-dihydroxyvitamin D₄ [1α,25-(OH)₂D₄]

[0064] The procedure of Example 3 is repeated using the active vitamin Danalogue 1α,25-(OH)₂D₄, and the specific binding is determined. Theresults demonstrate that 1α,25-(OH)₂D₄ has strong affinity for prostateVDR, indicating that 1α,25-(OH)₂D₄ has potent biological activity inrespect of prostate cells.

Gene Expression EXAMPLE 6

[0065] 1α,24-dihydroxy vitamin D₄ [1α,24-(OH)₂D₄]

[0066] Using the plasmids p(CT4)⁴TKGH, a vitamin D receptor(VDR)-expressing plasmid, and pSG5-hVDR1/3, a plasmid containing aGrowth Hormone (GH) gene, under the control of a vitamin D-responsiveelement (VDRE), experiments were conducted to explore the ability of1α,24-(OH)₂D₄ to induce vitamin D-dependent growth hormone acting as areporter gene compared to that of 1α,25-(OH)₂D₃. Cells in culture weretransfected with these two plasmids. One plasmid contained the gene forGrowth Hormone (GH) under the control of the vitamin D responsiveelement (VDRE) and the other plasmid contained the structural gene forthe vitamin D receptor (VDR). These transfected cultures were incubatedwith 1α,24-(OH)₂D₄ or 1α,25-(OH)₂D₃, and the production of growthhormone was measured. Table 2 below shows the results of this assay:TABLE 2 Induction of Growth Hormone by Vitamin D Compounds ConcentrationGrowth Hormone Compound Used (M) Induction (ng/ml) 1,25-(OH)₂D₃ 1 ×10⁻¹⁰ 39 1,25-(OH)₂D₃ 5 × 10⁻¹⁰ 248 1,24-(OH)₂D₄ 5 × 10⁻¹⁰ 1651,24-(OH)₂D₄ 1 × 10⁻⁹  628 1,24-(OH)₂D₄ 5 × 10⁻⁹  1098

[0067] These data show that the ability of 1α,24-(OH)₂D₄ to stimulatevitamin D-dependent growth hormone is nearly equivalent to that of1α,25-(OH)₂D₃. Such results are truly surprising and would not have beenexpected by following the teachings of the prior art.

EXAMPLE 7

[0068] 1α,24(S)-dihydroxyvitamin D₂ and 1α,24(R)-dihydroxyvitamin D₂[1α,24(S)-(OH)₂D₂ and 1α,24(R)-(OH)₂D₂]

[0069] The gene expression study described in Example 6 was conducted tocompare the biological activity in vitro of chemically synthesized1α,24(S)-(OH)₂D₂ and 1α,24(R)-(OH)₂D₂, with 1α,25-(OH)₂D₃ and 25-OH-D₃.The vitamin D-dependent transcriptional activation model system was usedin which plasmids pSG5-hVDR1/3 and p(CT4)⁴TKGH were co-transfected intoGreen monkey kidney, COS-1 cells.

[0070] Transfected cells were incubated with vitamin D metabolites andgrowth hormone production was measured. As shown in Table 3, both1α,24(S)-(OH)₂D₂ and its epimer, 1α,24(R)-(OH)₂D₂, had significantlymore activity in this system than 25-OH-D₃, with 1α,24(S)-(OH)₂D₂ havingnearly the same activity as 1α,25-(OH)₂D₃. TABLE 3 Vitamin D-InducibleGrowth Hormone Production In Transfected Cos-1 Cells Vitamin D-Inducible Growth Hormone Production Net vitamin Total GH D-inducibleMolar Production* GH-production Inducer Concentration (ng/ml) (ng/ml)Ethanol 44 0 25-OH-D₃ 1 × 10⁻⁷  245 201 1 × 10⁻⁶  1100 1056 1 × 10⁻⁵ 775 731 1α,25-(OH)₂D₃ 1 × 10⁻¹⁰ 74 30 1 × 10⁻⁹  925 881 l × 10⁻⁸  14751441 1α,24(S)-(OH)₂D₂ 5 × 10⁻¹⁰ 425 381 5 × 10⁻⁹  1350 1306 5 × 10⁻⁸ 1182 1138 1α,24(R)-(OH)₂D₂ 1 × 10⁻⁹  80 36 1 × 10⁻⁸  1100 1056 1 × 10⁻⁷ 1300 1256

Inhibition of Prostrate Cell Proliferation EXAMPLE 8

[0071] 1α,24-dihydroxyvitamin D₂ [1α,24-(OH)₂D₂]

[0072] Inhibition of cell proliferation is demonstrated using thetechniques of Skowronski et al., 132 Endocrinology (1993) 1952-1960 and136 Endocrinology (1995) 20-26, both of which are incorporated herein byreference. The cell lines, LNCaP and PC-3, which are derived from humanprostate adenocarcinoma, are seeded in six-well tissue culture plates ata density of about 50,000 cells/plate. After the cells have attached andstabilized, about 2-3 days, the medium is replenished with mediumcontaining vehicle or the active vitamin D analogue 1α,24-(OH)₂D₂, atconcentrations from 10-11 M to 10-7 M. Medium containing test analogueor vehicle is replaced every three days. After 6-7 days, the medium isremoved, the cells are rinsed, precipitated with cold 5% trichloroaceticacid, and washed with cold ethanol. The cells are solubilized with 0.2 Nsodium hydroxide, and the amount of DNA determined by standardprocedures. The results show that cultures incubated with 1α,24-(OH)₂D₂in accordance with the present invention have significantly fewer cellsthan the control cultures.

EXAMPLE 9

[0073] 1α,24-dihydroxy vitamin D₄ [1α,24-(OH)₂D₄]

[0074] The procedure of Example 8 is repeated using the active vitamin Danalogue 1α,24-(OH)₂D₄, and the cell number is determined. Culturesincubated with 1α,24-(OH)₂D₄ have significantly fewer cells than thecontrol cultures.

EXAMPLE 10

[0075] 1α,25-dihydroxyvitamin D₄ [1α,25-(OH)₂D₄]

[0076] The procedure of Example 8 is repeated using the active vitamin Danalogue 1α,25-(OH)₂D₄, and the cell number is determined. Culturesincubated with 1α,25-(OH)₂D₄ have significantly fewer cells than thecontrol cultures.

Stimulation of Prostate Cell Differentiation EXAMPLE 11

[0077] 1α,24-dihydroxyvitamin D₂ [1α,24-(OH)₂D₂]

[0078] Using the techniques of Skowronski et al., 132 Endocrinology(1993) 1952-1960 and 136 Endocrinology (1995) 20-26, both of which areincorporated herein by reference, cells of the cell line, LNCaP, whichis derived from a human metastatic prostate adenocarcinoma and known toexpress PSA, are seeded in six-well tissue culture plates at a densityof about 50,000 cells/plate. After the cells have attached andstabilized, about 2-3 days, the medium is replenished with mediumcontaining vehicle or the active vitamin D analogue, 1α,24-(OH)₂D₂, atconcentrations from 10⁻¹¹ M to 10⁻⁷ M. After 6-7 days, the medium isremoved and stored at −20° C. for prostate specific antigen (PSA)analysis.

[0079] The cells from parallel cultures are rinsed, precipitated, andthe amount of DNA determined by standard procedures. PSA is measured bystandard known methods. Cultures incubated with 1α,24-(OH)₂D₂ havesignificantly more PSA than control cultures when expressed as mass ofPSA/cell.

EXAMPLE 1.2

[0080] 1α,24-dihydroxyvitamin D₄ [1α,24-(OH)₂D₄]

[0081] The procedure of Example 12 is repeated except the active vitaminD analogue is 1α,24-(OH)₂D₄. The PSA is measured and cultures incubatedwith 1α,24-(OH)₂D₄ have significantly more PSA than control cultureswhen expressed as mass of PSA/cell.

EXAMPLE 13

[0082] 1α,25-dihydroxyvitamin D₄ [1α,24-(OH)₂D₄]

[0083] The procedure of Example 12 is repeated except the active vitaminD analogue is 1α,25-(OH)₂D₄. The PSA is measured and cultures incubatedwith 1α,25-(OH)₂D₄ have significantly more PSA than control cultureswhen expressed as mass of PSA/cell.

Clinical Studies EXAMPLE 14

[0084] 1α,24-dihydroxy vitamin D₂ [1α,24-(OH)₂D₂]

[0085] Patients with advanced androgen-independent prostate cancerparticipate in an open-labeled study of 1α,24-(OH)₂D₂. Qualifiedpatients are at least 40 years old, exhibit histologic evidence ofadenocarcinoma of the prostate, and present with progressive diseasewhich had previously responded to hormonal intervention(s). On admissionto the study, patients begin a course of therapy with oral 1α,24-(OH)₂D₂lasting 26 weeks, while discontinuing any previous use of calciumsupplements, vitamin D supplements, and vitamin D hormone replacementtherapies. During treatment, the patients are monitored at regularintervals for: (1) hypercalcemia, hyperphosphatemia, hypercalciuria,hyperphosphaturia and other toxicity; (2) evidence of changes in theprogression of metastatic disease; and (3) compliance with theprescribed test drug dosage.

[0086] The study is conducted in two phases. During the first phase, themaximal tolerated dosage (MTD) of daily oral 1α,24-(OH)₂D₂ is determinedby administering progressively higher dosages to successive groups ofpatients. All doses are administered in the morning before breakfast.The first group of patients is treated with 25.0 pg of 1α,24-(OH)₂D₂.Subsequent groups of patients are treated with 50.0, 75.0 and 100.0μg/day. Dosing is continued uninterrupted for the duration of the studyunless serum calcium exceeds 11.6 mg/dL, or other toxicity of grade 3 or4 is observed, in which case dosing is held in abeyance until resolutionof the observed toxic effect(s) and then resumed at a level which hasbeen decreased by 10.0 μg.

[0087] Results from the first phase of the study show that the MTD for1α,24-(OH)₂D₂ is above 20.0 μg/day, a level which is 10- to 40-foldhigher than can be achieved with 1α,25-(OH)₂D₃. Analysis of bloodsamples collected at regular intervals from the participating patientsreveal that the levels of circulating 1α,24-(OH)₂D₂ increaseproportionately with the dosage administered, rising to maximum levelswell above 100 pg/mL at the highest dosages, and that circulating levelsof 1α,25-(OH)₂D₃ are suppressed, often to undetectable levels. Serum andurine calcium are elevated in a dose responsive manner. Patients treatedwith the MTD of 1α,24-(OH)₂D₂ for at least six months report that bonepain associated with metastatic disease is significantly diminished.

[0088] During the second phase, patients are treated with 1α,24-(OH)₂D₂for 24 months at 0.5 and 1.0 times the MTD. After one and two years oftreatment, CAT scans, X-rays and bone scans used for evaluating theprogression of metastatic disease show stable disease or partialremission in many patients treated at the lower dosage, and stabledisease and partial or complete remission in many patients treated atthe higher dosage.

EXAMPLE 15

[0089] 1α-hydroxyvitamin D₂ [1α-OH-D₂]

[0090] The study of Example 14 is repeated for the active vitamin Dcompound, 1α-OH-D₂. The results of the phase one study indicate thatpatients treated with the MTD of 1α-OH-D₂ for at least six months reportthat bone pain associated with metastatic disease is significantlydiminished. The results of the phase two study indicate that after twoyears, CAT scans, X-rays and bone scans used for evaluating theprogression of metastatic disease show stable disease or partialremission in many patients treated at the lower dosage, and stabledisease and partial or complete remission in many patients treated atthe higher dosage.

[0091] While the present invention has now been described andexemplified with some specificity, those skilled in the art willappreciate the various modifications, including variations, additions,and omissions, that may be made in what has been described. Accordingly,it is intended that these modifications also be encompassed by thepresent invention and that the scope of the present invention be limitedsolely by the broadest interpretation lawfully accorded the appendedclaims.

1. A method of inhibiting the hyperproliferative activity of humanprostatic neoplastic or hyperplastic cells, comprising treating thecells with an effective amount of a 1α-hydroxyvitamin D compound havinga hydrocarbon moiety substituted at C-24.
 2. The method of claim 1,wherein said 1α-hydroxvitamin D compound is represented by formula (I)

wherein B and C each are hydrogen or a carbon-carbon bond, thus forminga double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl group, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes abond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or,taken with R¹ or R², constitutes a double bond.
 3. The method of claim2, wherein the compound of formula (I) is 1α,24-dihydroxyvitamin D₂,1α,24-dihydroxyvitamin D₄, 1α,25-dihydroxyvitamin D₂,1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitamin D₂ or 1α-hydroxyvitamin D₄.4. The method of claim 1, wherein said treating step includes inhibitingproliferation of, and inducing and enhancing differentiation in saidprostatic cells.
 5. A method for the treatment of prostatic diseasescharacterized by abnormal cell differentiation or cell proliferation,comprising administering to a male human or animal in need of suchtreatment an effective proliferation-inhibiting amount of a compound offormula (I)

wherein B and C each are hydrogen or a carbon-carbon bond, thus forminga double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl group, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes abond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or,taken with R¹ or R², constitutes a double bond.
 6. The method of claim5, wherein said therapeutic amount is 0.01 μg/kg/day to 2.0 μg/kg/day.7. A method of treating human prostate cancer, comprising administeringto a male subject, who has prostate cancer, an effective amount of afirst anticancer agent which is an active vitamin D compound to decreaseor stabilize the cellular abnormal proliferative activity of the cancer,said compound or its in vivo metabolite having a VDR binding affinitysubstantially equivalent to the binding affinity of1α,25-dihydroxyvitamin D₃ and a hypercalcemia risk substantially lowerthan that of 1α,25-dihydroxyvitamin D₃.
 8. The method of claim 7,wherein said active vitamin D is administered in a mixture including asecond anticancer agent selected from the group consisting ofestramustine phosphate, prednimustine, cisplatin, 5-fluoro-uracil,melphalan, hydroxyurea, mitomycin, idarubicin, methotrexate, adriamycinand daunomycin.
 9. A pharmaceutical composition, comprising (a) a firstanticancer agent which is an active vitamin D compound, and (b) an agentselected from the group consisting of (i) a second anticancer agent,(ii) a bone agent, (iii) an androgen control agent and (iv) a5α-reductase inhibitor and combinations thereof.
 10. The pharmaceuticalcomposition of claim 9, wherein said active vitamin D compound isrepresented by formula (I)

wherein B and C each are hydrogen or a carbon-carbon bond, thus forminga double bond between C-22 and C-23; R¹ and R² are identical ordifferent and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl,O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl,O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R¹and R² cannot both be an alkenyl group, or taken together with thecarbon to which they are bonded, form a C₃-C₈ cyclocarbon ring; R³ islower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl,O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lowercycloalkyl; X¹ is hydrogen or hydroxyl, or, taken with R³, constitutes abond when R³ is an alkenyl group, and X² is hydrogen or hydroxyl, or,taken with R¹ or R², constitutes a double bond.
 11. The pharmaceuticalcomposition of claim 9, wherein active vitamin D compound is selectedfrom the group consisting of 1α,24-dihydroxyvitamin D₂,1α,24-dihydroxyvitamin D₄, 1α, 25-dihydroxyvitamin D₂,1α,25-dihydroxyvitamin D₄, 1α-hydroxyvitamin D₂ or 1α-hydroxyvitamin D₄.12. The pharmaceutical composition of claim 9, wherein said secondanticancer agent is selected from the group consisting of estramustinephosphate, prednimustine, cisplatin, 5-fluoro-uracil, melphalan,hydroxyurea, mitomycin, idarubicin, methotrexate, adriamycin anddaunomycin.
 13. The pharmaceutical composition of claim 9, wherein saidactive vitamin D compound is present in a dosage range of about 0.01μg/kg/day to about 2.0 μg/kg/day.
 14. The pharmaceutical composition ofclaim 10 further including a pharmaceutically acceptable carrier. 15.The pharmaceutical composition of claim 9, wherein said androgen controlagent is selected from the group consisting of an estrogen, LHRHanalogue, an antiestrogen and an antiandrogen.
 16. The pharmaceuticalcomposition of claim 9, wherein said a 5α-reductase enzyme inhibitor isfinasteride.
 17. The pharmaceutical composition of claim 9, wherein saidbone agent is selected from the group consisting of conjugatedestrogens, antiestrogens, calcitonin, sodium fluoride, bisphosphonate,calcium supplements, cobalamin, pertussis toxin and boron.
 18. A methodof treating a human to alleviate the hyperproliferative cellularactivity of prostatic cancer or hyperplasia, comprising administering toa human in need thereof a therapeutically effective amount of an activevitamin D compound having a hydrocarbon moiety substituted at C-24.